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IL-13 antibody and preparation method and use thereof

a technology of il-13 and antibody, applied in the field of antibodies, can solve the problems of high mortality, high mortality, and the inability to relieve patients by conventional airway inhalation, and achieve the effects of inhibiting il-13-induced expression, high affinity, and inhibiting the secretion

Active Publication Date: 2021-10-05
SHANGHAI PHARMAEXPLORER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides an IL-13 antibody with high-affinity for its protein and can significantly inhibit the functions of IL-13, such as its secretion and expression of target genes. The antibody can also prevent symptomatic bronchial asthma in mice induced by IL-13. The protein of the invention can be used in the preparation of a medicine for preventing or treating bronchial asthma.

Problems solved by technology

However, about 10% of patients cannot be relieved by conventional airway inhalation.
These patients usually need oral steroid drugs with serious side effects to control the condition, which are still accompanied by high mortality.
Not only is the quality of life of patients greatly affected, but the direct medical costs and indirect costs result in huge individual and the socio-economic burdens.

Method used

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  • IL-13 antibody and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

embodiment 1

[0122]Expression and Purification of Human IL-13 and Human IL-13R130Q Variants

[0123]A nucleotide encoding six histidines was added to the 3′ end of the nucleotide sequence encoding Met1-Asn132 in the amino acid sequence of human IL-13 protein (see GenBank database, accession number: AAK53823.1) to obtain a nucleotide sequence of the recombinant human IL-13 protein with his tag (as shown in SEQ ID No. 61 of the Sequence Listing).

[0124]Alternatively, a nucleotide encoding six histidines was added to the 3′ end of the nucleotide sequence encoding Met1-Asn132 in the amino acid sequence of human IL-13 protein variant IL-13 R130Q to obtain the nucleotide sequence of the recombinant human IL-13 R130Q variant with his tag (as shown in SEQ ID No. 62 of the Sequence Listing).

[0125]The nucleotide sequence encoding the recombinant human IL-13 protein with his tag and the nucleotide sequence of the recombinant human IL-13R130Q variant with his tag were cloned into a PCP vector (purchased from In...

embodiment 2

[0131]Obtain Lead Antibody by Phage Technology

[0132]1. Biotin Labeling of Immunogen A

[0133]Biotin-X-X-NHS (purchased from Sigma Aldrich) and the purified immunogen A prepared in embodiment 1 (hBLyS-ECD) were mixed at a molar ratio of 3:1 and stood for 30 minutes at room temperature. Subsequently, 50 mM final concentration of 1 M NH4Cl was pipetted to terminate the reaction and a biotin-labeled immunogen A was obtained. Later, the biotin-labeled immunogen A was then dialyzed in PBS phosphate buffer (pH 7.4). At last, broadford reagent (purchased from Pierce) was used and the concentration was determined using BSA as a standard sample (for details, see Bradford, 1976, Anal Biochem 72: 248-54). The experimental results are shown in Table 4. The OD595 nm value of the biotin-labeled immunogen A is determined as 0.49, and the concentration of biotin-labeled immunogen A is calculated to be 0.319 mg / mL according to the curve fitted by the standard sample.

[0134]

TABLE 4Determination of the co...

embodiment 3

[0143]Obtain Lead Antibody Using Hybridoma Technology

[0144]1. Mouse Immunization

[0145]Immunogen A was used to immunize Balb / c, SJL / J mice (supplied by Shanghai SLAC Laboratory Animal Co., Ltd.) aged 6-8 weeks. Mice were feed under Specific pathogen Free (SPF) conditions after received. The initial immunization dose was 50 μg of immunogen A per mouse. The protein was emulsified in Freund's complete adjuvant and 0.25 mL of which was injected subcutaneously into the tail. 2 weeks after the initial immunization, the immunization was boosted. Immunogen A (25 μg per mouse) was emulsified in Freund's incomplete adjuvant and 0.25 mL of which was intraperitoneally injected. The interval of each booster immunization was three weeks. Serum samples were collected one week after each booster immunization, and the antibody titer in the serum was tested by ELISA and the activity of the antibody in the serum was measured by receptor-ligand binding-inhibition assay. Mice with higher serum titers and...

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Abstract

The present invention discloses IL-13 antibody, method of its preparation and use thereof. The IL-13 antibody comprises one or more of heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of heavy chain variable region of the IL-13 antibody, and / or one or more of light chain CDR1, light chain CDR2, and light chain CDR3 of light chain variable region of the IL-13 antibody. The IL-13 antibody has a high affinity and can significantly inhibit the secretion of thymus activation-regulated chemokine and periostin as well as the expression of vascular cell adhesion molecule-1 induced by IL-13, it can significantly inhibit airway hyperresponsiveness in mice induced by IL-13, and therefore can be used in the preparation of drugs for preventing or treating IL-13 expression or dysfunction related diseases.

Description

[0001]This application claims priority of Chinese Patent Application No. 201610333020.X filed on May 18, 2016 and Chinese Patent Application No. 201610474103.0 filed on Jun. 14, 2016. The entire content of the aforementioned application is hereby incorporated by reference.FIELD OF INVENTION[0002]The present invention relates to the antibody, and more specifically, relates to IL-13 antibody, method of its preparation and use thereof.PRIOR ARTS[0003]Bronchial asthma (asthma for short) is a common chronic airway inflammatory disease. It is usually accompanied by increased airway responsiveness, and recurrent symptoms such as wheezing, shortness of breath, chest tightness and / or coughing. Since the 1970s, asthma has become widespread. By 2011, around 235 million to 300 million people were affected worldwide, and about 250,000 people lost their lives. Clinically, asthma is usually treated by inhaling glucocorticoids such as steroids and assisting long-acting beta-adrenergic receptor agon...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K16/24A61K39/395G01N33/68C12N15/85C07K16/06A61K39/00
CPCC07K16/244A61K39/395C07K16/065C07K16/24C12N15/85G01N33/68G01N33/6869A61K2039/505C07K2317/56C07K2317/565C07K2317/76C12N2015/8518G01N2333/5437C07K2317/24C07K2317/92C07K2317/622C07K2317/21A61P11/00
Inventor YANG, YIZHENGONG, SHIYONGHAO, LIJUANWU, JIANYANG, XINXIUZHONG, QINHU, SHAOPINGLEUNG, STEWARTDUAN, QINGLIU, LILE
Owner SHANGHAI PHARMAEXPLORER
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